Effect of ionic liquid on the enzymatic synthesis of cello-oligosaccharides and their assembly into cellulose materials

Carbohydrate Polymers 301 (2023) 120302

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effect of ionic liquid on the enzymatic synthesis of cello-oligosaccharides
and their assembly into cellulose materials
Chao Zhong a, Krisztina Zajki-Zechmeister a, Bernd Nidetzky a, b, *
a
b

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, 8010 Graz, Austria
Austrian Centre of Industrial Biotechnology (acib), 8010 Graz, Austria

A R T I C L E I N F O

A B S T R A C T

Keywords:
Synthetic cellulose
Ionic liquid
Chain self-assembly
Hydrogel
Phosphorylase bio-catalysis

Imidazolium-based ionic liquids are important solvents for the processing of natural cellulose. Little is known
about their use in synthesizing cellulose via bottom-up polymerization of β-1,4-D-glucosyl chains in solution.
Here, we analyzed cellodextrin phosphorylase-catalyzed synthesis of cello-oligosaccharides, and the subsequent
spontaneous self-assembly of the chains, in the presence of cellulose-dissolving ionic liquid, 1,3-dimethylimida­

zolium dimethyl phosphate ([Dmim]DMP) or 1-ethyl-3-methylimidazolium acetate ([Emim]OAc). The average
chain length dropped from ~7.4 in buffer to ~6.4 in ionic liquid (30 vol%). The synthetic cellulose exhibited
allomorph II crystal structure and showed nanosheet morphology of 4–5 nm thickness and several μm length. Its
suspensions were hydrogels with viscoelastic properties dependent on solvent conditions used. Reactions in 10
vol% [Dmim]DMP or [Emim]OAc gave a hydrogel with elastic modulus of ~13 kPa and loss factor of ~0.18.
Collectively, interactions of the ionic liquid with enzyme and cello-oligosaccharides delimit the polymerization
and tune the assembly into cellulose networks.

1. Introduction
Cellulose is an abundant and eco-friendly natural polymer composed
of β-1,4-linked D-glucose units. The production of cellulosic materials is
usually based on top-down processing of lignocellulosic biomasses (e.g.,
woody materials) (Abdul Khalil et al., 2014; Brinchi, Cotana, Fortunati,
& Kenny, 2013). This often involves mechanical/physical disruption of
the original chain structure in cellulose biomaterials (Abdul Khalil et al.,
2014) as well as partial chemical depolymerization of the poly­
saccharide chains (Brinchi et al., 2013). In the extent that top-down
processing alters the original cellulose structure physically and chemi­
cally (Abdul Khalil et al., 2014; Phanthong et al., 2018), bottom-up
synthesis of cellulose chains can present a promising alternative of cel­
lulose material production. The bottom-up concept involves synthetic
build-up of cellulose chains, which then self-assemble into a hierar­
chically organized material. Assembled structures of synthetic cellulose
chains have been prepared by different strategies (Habibi, Lucia, &
Rojas, 2010; Kontturi et al., 2018). The enzymatic approach of synthesis
is gaining increased attentions since it offers simplicity and flexibility in
controlling the properties of the resulting cello-oligomers and hence
their self-assembly into cellulose materials. Among the known options

for the enzymatic synthesis of cellulose (Hiraishi et al., 2009; Petrovic,

ănen et al., 2020; Serizawa,
Kok, Woortman, Ciric, & Loos, 2015; Pylkka
Kato, Okura, Sawada, & Wada, 2016), the approach using cellodextrin
phosphorylase (CdP, EC 2.4.1.49) is promising, given the high chemical
purity of the products, the simple substrates used, and the flexibility to
prepare reducing end-functionalized cello-oligomers (Bulmer, de
Andrade, Field, & van Munster, 2021; Nakai, Kitaoka, Svensson, &
Ohtsubo, 2013). Cello-oligomers prepared by the CdP reaction selfassemble into different material structures in situ depending on the
conditions used (Hata & Serizawa, 2021; Nidetzky & Zhong, 2020;
Nigmatullin, de Andrade, Harniman, Field, & Eichhorn, 2021; Sugiura,
Sawada, Tanaka, & Serizawa, 2021).
Several studies of CdP-catalyzed synthesis of cello-oligosaccharides
have shown that bulk parameters (e.g., pH, temperature) can affect
the properties of the resulting cellulose (Hata, Kojima, Maeda, Sawada,
& Serizawa, 2020; Hata, Sawada, Marubayashi, Nojima, & Serizawa,
2019). In addition, polymers (Hata et al., 2017) and colloidal particles
(Hata, Sawada, Sakai, & Serizawa, 2018) that give a macromolecular
crowding effect or cause a viscosity increase also affect the enzymatic
synthesis and the subsequent self-assembly of the cello-oligomers.
Earlier works have placed a strong focus on the effect of

* Corresponding author at: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, 8010 Graz, Austria.
E-mail addresses: (C. Zhong), (K. Zajki-Zechmeister), (B. Nidetzky).
/>Received 24 August 2022; Received in revised form 21 October 2022; Accepted 31 October 2022
Available online 3 November 2022
0144-8617/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( />

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Carbohydrate Polymers 301 (2023) 120302

macromolecules (i.e., molecular mass of about 20 to 150 kDa) on the
enzymatic cellulose synthesis (Hata et al., 2017; Hata et al., 2018).
However, effects of small-molecule additives (< 1 kDa) have not been
fully explored up to now. A recent study reported the use of organic
solvents (e.g., dimethyl sulfoxide, ethanol) to prevent the precipitation
of the incipient cellulose oligomers during enzymatic synthesis (Hata,
Fukaya, Sawada, Nishiura, & Serizawa, 2019). It was suggested that the
cellulose chains might become more strongly solvated due to the in­
teractions (e.g., hydrogen bonding) with the solvents. The exciting idea
was put forward that small molecules could have a role in tuning the
assembly of the synthesized cello-oligomers (Hata, Fukaya, et al., 2019).
In the present study, therefore, the enzymatic synthesis of cellooligosaccharides was investigated using ionic liquids (ILs) as smallmolecule additives or co-solvents. The ionic liquids might affect both
the synthesis of the cello-oligosaccharides by the enzyme and the sub­
sequent self-assembly driven aggregation of the chains in bulk solution.
ILs are ion-containing organic salts that are known as one of the best
solvents for carbohydrate polymers including cellulose. ILs are effective
because they contain loosely bound ions that can interact with polar
groups (e.g., -OH) of oligo- and polysaccharides thus to solubilize them
(Li, Wang, Liu, & Zhang, 2018; Morais et al., 2020; Verma et al., 2019;
Wang, Gurau, & Rogers, 2012). To this end, 1,3-dimethylimidazolium
dimethyl phosphate ([Dmim]DMP) and 1-ethyl-3-methylimidazolium
acetate ([Emim]OAc) were selected for their remarkable cellulose
dissolution capacities (Koide, Urakawa, Kajiwara, Rosenau, & Wataoka,
2020; Li et al., 2018; Lopes, Bermejo, Martín, & Cocero, 2017; Zheng,
Harris, Bhatia, & Thomas, 2019). We hypothesized that the ILs could
interact with the synthesized cello-oligomers mainly through hydrogen
bonds that might delimit the oligomerization and alter product prop­
erties accordingly. The CdP-catalyzed reactions were carried out in the
presence of IL (~30% by volume), and an increased soluble yield of

cellulose (by ~40%) was observed when ILs were used. Solid products
were structurally analyzed (i.e., degree of polymerization (DP),
morphology, crystallinity) to reveal the effect of ILs on the assynthesized cellulose. In addition, hydrogels with viscoelastic proper­
ties were obtained depending on the conditions used. It was hypothe­
sized that the interactions with IL facilitate the assembly of cellulose into
highly-ordered material networks. Overall, this study contributes to a
better understanding of the role of small molecules in the enzymatic
synthesis of oligo- and polysaccharides.

2.3. Oligomerization reaction
Reactions (in 0.5 mL volume) were performed at 45 ◦ C and 300 rpm
through incubation on a ThermoMixer C (Eppendorf, Vienna, Austria)
for 24 h. α-D-Glucose 1-phosphate (αGlc1-P, 150 mM) and cellobiose (10
mM) were incubated with CcCdP (0.5, 1.0, and 2.0 U/mL, buffer ac­
tivity) in 50 mM MES buffer (pH 7.0) containing ionic liquid ([Dmim]
DMP or [Emim]OAc) in a volume fraction of 10, 20, 30, and 40%,
respectively. The control reaction was performed under exactly the same
conditions but without ionic liquid.
Insoluble materials were generated during the reactions. To recover
the solid materials, reaction mixtures were centrifuged at 21,130 ×g for
at least 5 min (Centrifuge 5424/R, Eppendorf, Germany) until the su­
pernatant was clear. After removal of the supernatant, the pelleted
material was thoroughly resuspended in 1 mL of distilled water and then
centrifuged again at 21,130 ×g for 5 min (Centrifuge 5424/R, Eppen­
dorf). The washing step was repeated 3 times. In these steps, the su­
pernatant was carefully removed with a pipette, with the tips away from
the pellet to avoid loss of the material. The solid thus obtained was
lyophilized and then weighed. The insoluble ratio of the products was
defined as the molar ratio of glucosyl units in the solid (estimated from
the total amounts of insoluble products and the average DP of products

calculated from mass spectrometry analysis) to the glucosyl units
transferred from αGlc1-P during the reaction.
In addition, the supernatant of the reaction was heated (95 ◦ C, 5 min)
to inactivate the enzyme and then centrifuged. The conversion of αGlc1P was determined by the phosphate released into the supernatant.
Phosphate was measured by a colorimetric assay (Saheki, Takeda, &
Shimazu, 1985). Note that [Emim]OAc had no effect on the colorimetric
assay and the influence of [Dmim]DMP (i.e., intrinsic phosphate con­
tent) was eliminated from the assay.
2.4. Material characterization
2.4.1. Atomic force microscopy (AFM)
The measurement was performed at room temperature using a
Dimension FastScan Bio instrument (Bruker AXS, Karlsruhe, Germany)
equipped with a NanoScope V controller in tapping mode. Cellulose
(washed pellets) dispersed in water (~2 mg/mL, 60 μL) was loaded onto
a freshly cleaved mica surface and air dried. A FastScan-A probe (Bruker
AXS, Camarillo, USA) was used. Analysis was performed using Gwyd­
dion 2.55 ( />
2. Material and methods
2.1. Materials

2.4.2. Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF-MS)
Cellulose (washed pellets) suspended in water (~5 mg/mL) was
prepared for measurement, which was performed according to the
method described (Zhong, Zajki-Zechmeister, & Nidetzky, 2021). Mass
spectra analysis was done using the mMass ( />The number-average molecular weight (Mn) was calculated using the
∑
∑
relationship, Mn = i (Ni × Mi)/ i Ni, where Ni is the peak intensity of
the i-th cello-oligomer species and Mi is the molar mass of that species.

Here, sodium and potassium ion adducts of the oligomer in each DP (m/z
+23 and +39) were included. The average DP was calculated using the
relationship, DP = (Mn − 18)/Mo, where Mo is the molecular mass of
dehydrated glucose (in cellulose), 162.2 Da (Petrovic et al., 2015).

Unless stated otherwise, the chemicals used were of highest purity
available at Sigma-Aldrich (Vienna, Austria) or Carl Roth (Karlsruhe,
Germany). Ionic liquids, [Dmim]DMP and [Emim]OAc, were from abcr
GmbH (Karlsruhe, Germany).
2.2. Enzyme
Cellodextrin phosphorylase from Clostridium cellulosi (CcCdP; Gen­
Bank identifier CDZ24361.1) was prepared according to the methods
described (Zhong, Luley-Goedl, & Nidetzky, 2019). Briefly, enzyme was
expressed in Escherichia coli BL21(DE3) and purified via its N-terminal
His-tag. Enzyme stock solutions (20 mg/mL) in 50 mM MES buffer (pH
7.0) were stored at − 20 ◦ C without appreciable loss of activity for at
least one month. The stock solutions were used as single-use aliquots and
diluted to the desired working concentrations. The enzyme showed a
synthesis activity of 13.3 U/mg (on the acceptor substrate cellobiose) at
45 ◦ C in 50 mM MES buffer (pH 7.0) (Zhong et al., 2019; Zhong &
Nidetzky, 2022).

2.4.3. Proton nuclear magnetic resonance (1H NMR)
The 1H NMR spectra of the lyophilized material dissolved in 4%
NaOD-D2O (10 mg/mL) were recorded on a Varian Inova-500 NMR
spectrometer (Agilent Technologies, Santa Clara, CA, USA) using a
VNMRJ 2.2D software. The chemical shifts were recorded relative to
D2O (δH 4.8), and analyzed by MestReNova ().
The average DP of the product was calculated using the relationship, DP
= (H1 + Hα + Hβ)/(Hα + Hβ), where the H1, Hα and Hβ present the

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Carbohydrate Polymers 301 (2023) 120302

intensity of signals at 4.30 ppm (internal anomeric protons), 5.12 and
4.53 ppm (α- and β-anomeric proton), respectively (Hiraishi et al.,
2009).

synthesis of water-insoluble cellulose. However, no turbidity was
observed in the reactions with 40 vol% IL. The reaction in buffer without
IL gave a gel-like mixture that partially collapsed upon inversion
(Fig. 2a). The reactions containing 10–30 vol% IL also involved gel
formation (Fig. 2a), and a relatively stable gel was obtained in the re­
actions with 10 vol% IL (see later).
The αGlc1-P conversion (after 24 h) in the reactions decreased with
increasing IL concentration, from 56% in the control reaction to ~42%
in the reaction with 30 vol% IL (Fig. 3). A dramatic decline of αGlc1-P
conversion (to just ≤12%) was further observed in the reactions con­
taining 40 vol% IL. Time course analysis (Fig. 3) also revealed that the
αGlc1-P conversion was consistently lower when enzymatic reactions
were performed in the presence of IL. These results suggested that the ILs
caused a lowering of the apparent activity of the CcCdP. The inhibitory
effect on activity (hence, the reaction rate) was dependent on the IL
concentration used (Fig. 3). The IL additionally caused a decrease in
enzyme stability. Fig. 4a shows that incubation at IL concentrations of
≥20 vol% resulted in considerable loss of enzyme activity in 24 h. Note
that with the assay used, an irreversible process of enzyme inactivation

was measured. At 40 vol% of both [Dmim]DMP and [Emim]OAc, nearly
all of the original enzyme activity was lost. The two ILs used here are
ăm, Rovio, &
generally considered to be enzyme-friendly (Wahlstro
Suurnă
akki, 2012; H. Zhao, Jackson, Song, & Olubajo, 2006). Nonetheư
less, tolerance of CcCdP to them as co-solvents was limited.
There can be different reasons for enzyme inhibition in the presence
of IL (Zhao, 2005). One reason is a direct effect of the co-solvent on the
enzyme structure. The other is indirect and involves a co-solvent effect
on substrate accessibility to the enzyme (Endo, Hosomi, Fujii, Ninomiya,
& Takahashi, 2016; Li et al., 2018). The interaction of IL ions with the
carbohydrate substrates may change the substrate partitioning between
the solvent and the enzyme binding pocket, thus leading to a lowered
apparent affinity for substrate binding and thus a decreased activity. We
cannot distinguish between these possibilities based on the evidence
obtained. Nevertheless, the selected ILs were usable as co-solvents for
the purpose of current study when their concentrations were limited to
30 vol%.
Close inspection of the different reactions in Fig. 2 revealed that the
evolution of turbidity decreased with increasing concentration of each
IL. To quantitate the effect, the amount of insoluble product was
measured from each reaction (0.5 mL volume; N = 4). The mass of
insoluble product was 5.6 ± 0.2 mg from the buffer control reaction. It
was 4.8 ± 0.1 mg, 4.4 ± 0.4 mg and 3.2 ± 0.1 mg from the reaction in
the presence of 10, 20 and 30 vol% [Dmim]DMP, respectively. Using
[Emim]OAc, it was 4.9 ± 0.1 mg, 3.3 ± 0.2 mg and 1.8 ± 0.1 mg from
the reaction at 10, 20 and 30 vol%, respectively. The decrease in the
insoluble product formation dependent on the IL concentration used

2.4.4. X-ray diffraction (XRD)
Measurement of the lyophilized cellulose material was done ac­
cording to the method described (Zhong et al., 2021).
2.5. Rheological measurement
Dynamic rheological measurements were performed on a straincontrolled rheometer (MCR 502, Anton Paar, Austria) at 25 ◦ C, using
a cone-and-plate measurement geometry (CP 50-1) with 50 mm diam­
eter and 1◦ cone. For measurement, the sample (600 μL) was placed on
the Peltier plate. The linear viscoelastic range was measured with a
strain sweep (0.01–100%) at a fixed frequency of 10 rad/s. Frequency
sweeps were performed over an angular frequency range of 1–100 rad/s
with a constant strain amplitude of 0.1% (within the linear viscoelastic
range) to record the storage modulus (G′ ) and loss modulus (G′′ ) of the
mixtures.
3. Results and discussion
3.1. Enzymatic synthesis of cello-oligosaccharide chains in ILs
Our previous study of the bottom-up synthesis of cellooligosaccharides exploited the CdP-catalyzed reaction using cellobiose
as the “primer” substrate (Fig. 1a) (Zhong et al., 2019). The synthesized
cello-oligomers (with an average DP above 6) assembled into sheet-like
nanocelluloses that aggregated/precipitated from buffer solution (Kli­
macek, Zhong, & Nidetzky, 2021). Here, we hypothesized that due to
their known interaction with cellulose chains, ILs might tune the as­
sembly of the incipient cello-oligosaccharides and change the overall
properties of the synthetic cellulose materials.
We here showed a CcCdP-catalyzed synthesis of cellooligosaccharides in the presence of [Dmim]DMP or [Emim]OAc
(Fig. 1b). The two imidazolium-based ILs are well-known for their
cellulose-dissolving properties (Lopes et al., 2017; Wang et al., 2012).
Cello-oligosaccharides were synthesized at a donor/acceptor molar ratio
of 15:1. Earlier studies (Petrovic et al., 2015; Zhong et al., 2019) have
shown that donor/acceptor ratios as high as this favor the chain elon­
gation and so the formation of insoluble cellulose as the product. The

conditions used were thus adjusted to investigate the chain assembly in
the presence of IL. Reactions involving 2.0 U/mL CcCdP (buffer activity)
in the absence or presence of IL (10–40 vol%) were tested. The mixtures
with 0–30 vol% IL turned opaque after 24 h of reaction, indicating the

Fig. 1. Bottom-up enzymatic synthesis of cello-oligosaccharides in the presence of imidazolium-based IL. a) Reaction scheme of β-1,4-glycosylation of cellobiose
using αGlc1-P as the donor catalyzed by cellodextrin phosphorylase; b) Chemical structure of the imidazolium-based ILs used in the current study: [Dmim]DMP, 1,3dimethylimidazolium dimethyl phosphate; [Emim]OAc, 1-ethyl-3-methylimidazolium acetate.
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Carbohydrate Polymers 301 (2023) 120302

Fig. 2. Photographs of the reaction mixtures (after 24 h) at different enzyme activities. The activities here refer to the enzyme assay in buffer without IL. The
reactions were performed using 10 mM cellobiose, 150 mM αGlc1-P in 50 mM MES buffer (pH 7.0) containing IL concentrations of 0–30 vol% at 45 ◦ C, for 24 h. To
assess gelation, the tubes were inverted after the reactions.

Fig. 3. Time courses of αGlc1-P conversion from the enzymatic reactions with IL at various concentrations (0–30 vol%). a) [Dmim]DMP; b) [Emim]OAc. Reactions
using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL CcCdP (buffer activity) in 50 mM MES buffer (pH 7.0) containing IL at varied concentrations were performed at
45 ◦ C for 24 h.

might arise trivially from the fact that the conversion of the αGlc1-P
substrate was also lowered when the IL concentration was increased.
However, it might also involve a shift in the ratio of insoluble and sol­
uble products released in the enzymatic reaction when IL was present.
Fig. 4b shows that the portion of insoluble material in the total product
mass decreased dramatically (by up to 40%) as the IL concentration

increased. In buffer without IL, all of the product (≥98%) accumulated

in insoluble form. Ability of the IL to interact with the incipient cellooligosaccharides can arguably be related to the Kamlet-Taft parameter
of H-bond basicity (β). The β value of the two ILs used is ~1.0 while that
of water is only 0.18 (Lopes et al., 2017). Solvent interactions of the
cello-oligosaccharides that are stronger with the ILs than water could
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Carbohydrate Polymers 301 (2023) 120302

vol%), and it was slightly lower than that of the oligosaccharides syn­
thesized in the buffer control reaction (Table 1). This result was
consistent with the morphology analysis of the products (Fig. 6). AFM
observations of these sheet-like materials revealed an estimated thick­
ness of 4.9 ± 0.2 nm for the product from the control reaction. The
products from the [Dmim]DMP- and [Emim]OAc-containing reactions
(20 vol%) exhibited a lower thickness of 4.2 ± 0.2 nm and 4.1 ± 0.2 nm,
respectively. It was shown in the earlier works (Hata, Sawada, et al.,
2019; Serizawa, Fukaya, & Sawada, 2017) that the cellooligosaccharides are aligned perpendicular to the base plane of the
cellulose nanosheets. The nanosheet thickness is thus expected to reflect
the average DP of the synthetic cello-oligosaccharides.
Detailed analysis as summarized in Table 1 shows that the average
DP of the cello-oligosaccharide products decreased with increasing IL
concentration in the reactions. The result can arguably be explained by
the decrease of enzymatic reaction rate in the presence of the IL cosolvent. As mentioned before, the decrease in rate may involve direct
or indirect effect of the IL on the enzyme activity. The rate of chain
elongation in competition with the rate of chain aggregation delimits the
average DP of cello-oligosaccharide present in the insoluble cellulose
material. The relative portion of longer-chain cello-oligosaccharides

(DP ≥ 8) was dramatically reduced in the enzymatic reactions con­
taining IL (Fig. 5b). In agreement with these findings, the research of
Serizawa's group has shown that the average DP of cellulose chain can be
modulated by changing the volumetric CdP activity in the reaction
(Serizawa et al., 2017) or by using conditions (e.g., at lower temperature
20–30 ◦ C) that decrease the enzymatic rate (Hata, Fukaya, et al., 2019).
The cellulose materials were also analyzed by XRD. The XRD pat­
terns (Supplementary materials, Fig. S1) were identical for all the cel­
lulose materials, irrespective of the type of IL and the IL concentration
used in the synthetic reactions. The XRD peaks at 2θ of 12.5◦ , 20.1◦ , and
22.1◦ indicated a highly ordered cellulose material of allomorph II
crystal structure. The cellulose II is the most stable crystalline form of
cellulose (Moon, Martini, Nairn, Simonsen, & Youngblood, 2011) and is
typical of the cellulose materials prepared by self-assembly driven as­
sociation of cello-oligosaccharides from aqueous solution (Hata,
Sawada, et al., 2019; Hiraishi et al., 2009; Serizawa et al., 2017). Cel­
lulose II involves antiparallel organization of the cellulose chains. The
evidence from this study suggests that presence of the ILs used did not
alter the fundamental characteristics of crystalline cellulose formation
from the growing cello-oligosaccharide chains. It would appear there­
fore, that the IL effect was mostly on the enzymatic process of synthesis,
happening in solution.
In addition to the above features, no unassigned signal/peak was
detected in the NMR and MS spectra of the products from the enzymatic
reactions containing IL. This result suggested the absence of chemical
derivatization of the cello-oligosaccharides as-synthesized, consistent
with the notion that ILs engage in non-covalent interactions, mainly
involving hydrogen bonds and van der Waals forces, with oligo- and
polysaccharides (Verma et al., 2019).

Fig. 4. Effect of imidazolium-based IL on enzyme activity and insoluble
product formation in reactions catalyzed by CcCdP. a) Residual activity of
CcCdP after 24 h incubation in the buffer solutions containing [Dmim]DMP or
[Emim]OAc (0–40 vol%). The 50 mM MES buffer (pH 7.0) containing 2.0 U/mL
CcCdP (buffer activity) and varied IL concentrations (0–40 vol%) were incu­
bated at 45 ◦ C, for 24 h. The activity of CcCdP in solution after immediate
preparation (0 h) and 24 h incubation was measured, and the residual activity
was calculated; b) Insoluble ratio of cellulose products from the enzymatic re­
actions containing IL at varied concentrations. Reactions were performed using
10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL of CcCdP (buffer activity) in 50
mM MES buffer (pH 7.0) containing IL at 45 ◦ C, for 24 h.

explain the largely decreased tendency to undergo self-assembly driven
chain aggregation into solid material under conditions when the IL was
present. Addition of such IL can thus present a strategy to enhance the
soluble product release from the CdP-catalyzed reaction.

3.3. Gel-like properties of cellulose synthesized in the presence of IL
As mentioned above (see 3.1 Enzymatic synthesis of cellooligosaccharide chains in ILs), strong gelation behavior was observed
in reaction mixtures of enzymatic cellulose synthesis in the presence of
IL. While this behavior indicated the formation of network structures of
solid material in suspension, the underlying mechanisms are not well
understood from a number of earlier studies of gelation of cellulose in
the presence of IL (Hopson et al., 2021). In hydrogels prepared from
cellulose substrates that reflected different degrees of top-down pro­
cessing of natural raw materials, physical crosslinking by hydrogen
bonding represented the principal force of stable network formation
(Shen, Shamshina, Berton, Gurau, & Rogers, 2016). A series of experi­
ments were performed here to investigate the idea of a direct relation­
ship between the gel formation and the IL-mediated supramolecular

3.2. Structural characterization of the cello-oligosaccharides in insoluble
cellulose material
The chemical structure of the solid products was analyzed by 1H
NMR and MALDI-TOF mass spectrometry. The products, despite the
different reaction conditions used for synthesis, exhibited similar and
representative NMR signals (e.g., δH 4.3) assignable to the repeating
β-glucosyl units of cellulose (Fig. 5a) (Isogai, 1997). In addition, the
mass spectra, with the peak-to-peak mass difference of 162.2 Da (one
glucosyl unit), further confirmed the synthesis of cello-oligosaccharides
under these conditions (Fig. 5b). Here, the average DP of products
calculated from both the NMR and MS spectra was 6–7 for the cellooligosaccharides synthesized from the IL-containing reactions (10–30
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Fig. 5. Structural characterization of synthetic cellulose. a) 1H NMR and b) MALDI-TOF MS spectra of the solid products synthesized without and with IL at various
concentrations (D indicates [Dmim]DMP; and E indicates [Emim]OAc). Reactions were performed using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL CcCdP (buffer
activity) in 50 mM MES buffer (pH 7.0) containing IL at 45 ◦ C, for 24 h.

aforementioned reaction mixtures. The G′ was nearly frequencyindependent, and the G′′ was weakly frequency-dependent, having a
shallow minimum (0.5 U/mL reactions) or featuring decrease (1.0 and
2.0 U/mL reactions) at low frequency. This rheological behavior, also
observed in soft glassy materials (e.g., pastes and emulsions), might be
related to structural relaxations that occur between low and high fre­
quencies (Mason et al., 1997; Mendoza et al., 2018).
The G′ value is an indication of the hydrogel's ability to store

deformation energy in an elastic manner. It is correlated to the ability of
the material to revert to a solid state and to retain its shape after
cessation of shear (Ma et al., 2021). The G′ value normally increases
with increasing fiber material concentration, within certain range, in the
gel matrix, due to the stronger networks formed and the increased
contribution to stiffness (Ma et al., 2021; Mihranyan, Edsman, &
Strømme, 2007). This is suggested from the control reaction, where the
G′ increased from 7500 to 9600 Pa as the enzyme activity increased from
0.5 to 2.0 U/mL and accordingly the αGlc1-P utilization increased by
15% (Fig. 7b). Reactions at higher enzyme activity produced larger
amounts of cello-oligosaccharides, in particular at an early stage of the
conversion. The increased molecular crowding thus generated may have
promoted a stronger network of interactions between the initially
formed nuclei of insoluble cellulose (Hata et al., 2017; Korhonen &
Budtova, 2019). Interestingly, therefore, when IL co-solvent (10–20 vol
%) was used, the G′ declined at high enzyme activity. In reactions
containing [Dmim]DMP (10–20 vol%), the G′ value dropped by almost
80% as the enzyme activity increased from 0.5 to 2.0 U/mL. The effect of
varied enzyme activity was by far more significant on the resulting gel
properties than it was on the corresponding conversion of αGlc1-P,
which was changed by just 12% (Fig. 7b). Similarly, reactions in the
presence of [Emim]OAc (10–20 vol%) involved a decrease in the G′ by
almost 60% as the enzyme activity increased from 0.5 to 2.0 U/mL. The
corresponding change in αGlc1-P conversion was a mere 17% (Fig. 7b).
Moreover, reactions at 0.5 U/mL in the presence of 10 vol% [Dmim]
DMP or [Emim]OAc yielded cellulose hydrogels with a G′ of ~10–13
kPa (Fig. 7b) that was increased by up to 74% compared to the control (i.
e., hydrogel prepared in buffer lacking IL). Strikingly, these G′ values
were even higher than the G′ recorded for the control at 2.0 U/mL,
despite the fact that the control exhibited a 21% higher αGlc1-P con­

version than the reactions with 10 vol% IL. Survey of all the reactions

Table 1
Characterization of the solid cellulose products synthesized by CcCdP in the
presence of IL at varied concentrations.
Solvents

β value

Water/
buffer
[Dmim]
DMP

0.18

[Emim]
OAc

0.95b

1.0–1.1

a

Conc.
(vol%)

DP_1H
NMR

DP_MALDITOF MS

–

7.83

7.25

10

7.07

6.91

20

6.44

6.89

30
10

6.01
7.31

6.42
6.92

20

7.06

6.66

30

–

6.49

Allomorph
Cellulose
II
Cellulose
II
Cellulose
II
–
Cellulose
II
Cellulose
II
–

a

Ref. (Brandt, Hallett, Leak, Murphy, & Welton, 2010; Fukaya, Hayashi,
Wada, & Ohno, 2008).

b
Ref. (Zhang et al., 2012).

interactions between dispersed nanoscale nuclei of solid cellulose. The
volumetric enzyme activity was varied at three levels of 0.5, 1.0, and 2.0
U/mL (buffer activity), each at a variable IL content between 0 and 30
vol%. Gelation was observed in the majority of the reactions, except for
those at 0.5–1.0 U/mL in the presence of 30 vol% IL, which yielded a
suspension of the insoluble product (Fig. 2). The gel-like properties of
the reaction mixtures were further investigated by rheological means
(Mendoza, Batchelor, Tabor, & Garnier, 2018; Roy, Budtova, & Navard,
2003). In this respect, the storage modulus G′ describes the solid-like or
elastic behavior, and the loss modulus G′′ describes the liquid-like or
viscous behavior of the materials. The dependence of both G′ and G′′ on
the angular frequency (1–100 rad/s) was assessed. The behavior of the
mixtures was found to be predominantly elastic, indicated by the evi­
dence that G′ was larger than the corresponding G′′ over the entire fre­
quency range. Fig. 7a depicts this behavior for mixtures obtained from
the 0.5 U/mL reaction. Similar profiles of storage/loss modulus versus
angular frequency were obtained for the 1.0 and 2.0 U/mL reactions, as
shown in Figs. S2–3. The results confirmed the gel property of the
6


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Carbohydrate Polymers 301 (2023) 120302

Fig. 6. Atomic force microscopy (AFM) images of the synthesized cellulose: a) control; b) synthesized at 20 vol% [Dmim]DMP; c) synthesized at 20 vol% [Emim]
OAc. Materials were prepared from the reaction using 10 mM cellobiose, 150 mM αGlc1-P, 2.0 U/mL CcCdP (buffer activity) in 50 mM MES buffer (pH 7.0) with/

without IL at 45 ◦ C, for 24 h. The nanosheet crystalline material is shown, and its thickness (in single layer) was measured through cross-sectional analysis.

performed (Fig. 7b) suggested a minimum degree of αGlc1-P conversion
of ~42% required for gel formation. Reactions that yielded only sus­
pensions of insoluble cellulose (with G′ ≤ 1 kPa) exhibited low αGlc1-P
conversion of 27–36% (Figs. 2 and 7b). In summary, therefore, two
factors appear to be critical in order to promote cellulose gel formation
efficiently. A sufficient amount (concentration) of nanoscale cellulose
nuclei (supposedly in nanosheet form or smaller structure) must accu­
mulate as gel precursors from the synthesis reaction. Stiffness of the
resulting gel is affected by the precursor concentration. Physical cross­
linking of the gel precursors, and probably their further growth into
higher-order structures (e.g., nanoribbons; Hata, Fukaya, et al., 2019;
Hata, Sawada, et al., 2019), is then necessary to establish the final gel
network structure. The IL co-solvents appear to facilitate these processes
in particular and so generate a gel reinforcement effect. Fig. 8 illustrates
the proposed mode of cellulose gel formation under the assistance of IL.
Previous studies have demonstrated the transition into gels of cel­
lulose solutions in IL upon the addition of water. It was hypothesized
that the added water breaks some of the original cellulose-IL interactions
and thus promotes the restoration of cellulose-internal hydrogen
bonding interactions that lead to the stabilization of supramolecular
cellulose chain assemblies (Lee et al., 2017). With the formation of such
cellulose assemblies, able to interact with IL ions and serving as nuclei
for gelation via physical cross-linking, chain entanglement and forma­
tion of self-supporting gel networks could follow (Lee et al., 2017; Zhao
et al., 2020). Evidence of the current study emphasizes in particular the
prominent role of cellulose-IL interactions in the process of stable gel
formation. To allow for the relevant interactions with IL ions to be

developed in the pre-gelation state, a moderate synthetic rate that en­
ables self-assembly of cello-oligosaccharides of DP ≥ 6 is required. An
excessive synthetic rate can lead to a supramolecular aggregation of the
cellulose nuclei (chain assemblies) that may be too fast for IL ions to
intervene. Using an IL concentration (e.g., 10 vol%) suitably combined
with enzyme-catalyzed synthetic rate, the incipient cellooligosaccharides would self-assemble and generate cellulose nuclei
sufficiently stabilized/solvated by the IL ions in suspension (for a rele­
vant discussion of related effects of small molecules on cellulose as­
sembly, see Hata, Fukaya, et al., 2019). With IL ions mediating the
interactions among cellulose nuclei, they might be further entangled and
assembled, thus promoting the growth into a highly ordered matrix
(Fig. 8). This IL-mediated assembly route would impart a stronger
network of intermolecular interactions between the cellulose and confer
a higher elasticity to the resulting gel as compared to a gelation that
merely involves an all-cellulose network of interaction.
The viscoelastic properties of the gels were further evaluated on the
basis of the so-called loss factor (tan δ), defined as tan δ = G′′ /G′ . The tan
δ indicates how well the material performs in absorbing and dissipating
energy. Its value was found to increase with increasing IL concentration
used in the reactions (Fig. 9), suggesting a trend towards liquid-like
behavior of the semi-solid system (i.e., the viscous response is larger
than the elastic contribution) at the higher IL concentrations. The
observable trend (Fig. 9) might reflect the evidence discussed above that
the total content of insoluble cellulose in the suspension decreased as the
IL concentration was increased. A lowered concentration of cellulose
nuclei as gel precursors might affect the further crosslinking and so the
7


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Carbohydrate Polymers 301 (2023) 120302

Fig. 7. Rheological characterization of the synthetic
cellulose prepared in the presence of IL. a) Dynamic
viscoelastic properties of the cellulose product mix­
tures obtained from enzymatic reactions in the pres­
ence of IL (left panel, [Dmim]DMP; right panel,
[Emim]OAc). Reactions were done with 0.5 U/mL
(buffer activity). Storage modulus G′ and loss
modulus G′′ are shown with filled and open symbols,
respectively. Symbols: square (□) for control reac­
tion; circle (○) and triangle (△) for the reaction
containing 10 vol% and 20 vol% IL, respectively; b)
Heatmap of storage modulus G′ (upper panel) and
αGlc1-P donor conversion (lower panel) in the re­
actions with various enzyme loadings (0.5–2.0 U/mL)
and IL concentrations (0–30 vol%). Reactions with
enzyme loading (0.5, 1.0 and 2.0 U/mL of CcCdP,
buffer activity) were performed using 10 mM cello­
biose and 150 mM αGlc1-P in 50 mM MES buffer (pH
7.0) containing IL concentrations 0–30 vol% at 45 ◦ C,
for 24 h. The G′ values at angular frequency of 20
rad/s (middle range) were selected.

Fig. 8. Proposed mode of synthetic cellulose gel formation in the presence of IL co-solvent. Incipient cello-oligosaccharide chains self-assemble into nuclei of
insoluble cellulose, supposedly in nanosheet form. Direct intervention of the IL at this stage cannot be excluded but seems to be of minor importance. Under
involvement of IL, however, the cellulose nuclei are further assembled and grown into organized solid networks that confer gel-like properties to the resulting solidin-liquid dispersion (Hata, Fukaya, et al., 2019). Crosslinking of the cellulose nuclei involves participation from the IL ions via non-covalent hydrogen bonding.
Besides hydrogen bonds, the IL ions can interact via van der Waals and CH-π bonds (Liu, Sale, Holmes, Simmons, & Singh, 2010; Zhang et al., 2017);

resulting stiffness of material. A tan δ value of ≤1 is typical of concen­
trated polymer gels and one of ≤0.18 (phase angle δ ≤ 10◦ ) indicates a
strong gel (Mihranyan et al., 2007; Naji-Tabasi & Razavi, 2017). Mate­
rials from reactions at ≥20 vol% IL exhibited tan δ values of 0.24 or
greater (Fig. 9). These values are too high to be practically suitable for
hydrogel development. However, by controlling the IL content to ≤10
vol%, materials showing tan δ values of ≤0.18 were obtained. Such

materials can be classified as a truly elastic gel (Hopson et al., 2022;
Naji-Tabasi & Razavi, 2017). Taken together, the materials prepared at
10 vol% IL showed desirable rheological properties (i.e., a high G′ value
≥ 10 kPa; relatively low tan δ value) for hydrogel applications in gen­
eral. Such properties can be useful in further micro-structured fabrica­
tion by 3D printing (Ma et al., 2021). Also, the presence of ions in the
material (cellulose ionogel) can be important in electrochemical
8


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Carbohydrate Polymers 301 (2023) 120302

Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgement
This project has received funding from the European Union's Horizon

2020 research and innovation program under grant agreement No.
ărg Weber
761030 (CARBAFIN). The authors acknowledge Prof. Hansjo
and Prof. Brigitte Bitschnau from Graz University of Technology (TUG)
for the 1H NMR and XRD support, respectively. The authors also thank
Prof. Iain B.H. Wilson and Dr. Jorick Vanbeselaere from University of
Natural Resources and Life Sciences (Vienna) for MALDI-TOF MS sup­
port. Addition thanks to Prof. Michaela Flock and Dr. Angela Chemelli
from TUG for rheology measurement support.

Fig. 9. Viscoelastic properties of enzymatically synthesized cellulose gels. Loss
factor (tan δ) of the reaction mixtures as reflected by varied enzyme loadings
(0.5, 1.0, 2.0 U/mL, buffer activity) and IL concentrations (0–30 vol%): a)
[Dmim]DMP; b) [Emim]OAc. Symbols: square (□), diamond (⋄) and circle (○)
presents the reaction with enzyme loading of 0.5, 1.0 and 2.0 U/mL, respec­
tively. The loss factor was calculated using the values of G′ and G′′ at angular
frequency 20 rad/s (middle range).

Appendix A. Supplementary data

applications (Ge et al., 2021; Liu et al., 2020). Lastly, the hydrogels with
tunable properties have significant application potential in medical
treatment (Du et al., 2019; Shen et al., 2016).

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.carbpol.2022.120302.
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Using purified preparation of the CcCdP, bottom-up enzymatic syn­
thesis of cellulose material composed of short cello-oligosaccharide
chains (DP 6 - 7) was possible in the presence of water-miscible, cellu­
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or [Emim]OAc gave a robust material that showed elasticity and stiff­
ness desirable in hydrogel applications. Overall, the study expands the
scope of phosphorylase-catalyzed synthesis of cellulose materials and
advances the understanding of the role of IL co-solvent in cellulose selfassembly processes.
CRediT authorship contribution statement
Chao Zhong: Conceptualization, Methodology, Formal analysis,
Investigation, Writing – original draft, Visualization. Krisztina ZajkiZechmeister: Methodology, Investigation, Software. Bernd Nidetzky:
Conceptualization, Writing – review & editing, Resources, Funding
acquisition.
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